Antibodies immunologically specific for PD2, a protein that is amplified and overexpressed in pancreatic cancer

ABSTRACT

A human nucleic acid, PD2, its encoded protein and antibodies immunologically specific thereto are disclosed herein. The expression of the disclosed PD2 gene plays a key role in the regulation of differentiation and in the maintenance of the neoplastic state. The PD2 gene and its encoded protein represent valuable therapeutic targets in the differential diagnosis and therapy of pancreatic adenocarcinomas.

This application is a Divisional of U.S. patent application Ser. No.09/647,143, filed Sep. 27, 2000 now U.S. Pat. No. 6,680,196 which is a§371 application of PCT/US99/06633, filed Mar. 26, 1999 which in turnclaims priority under 35 U.S.C. §119(e) to US Provisional ApplicationNo. 60/079,649 filed Mar. 27, 1998. The entire disclosures of each ofthe above-identified applications is incorporated by reference herein.

Pursuant to 35 U.S.C. §202(c) it is acknowledged that the U.S.Government has certain rights in the invention described herein, whichwas made in part with funds from the National Institutes of Health,Grant Numbers: RO1 DK 46589, RO1 CA 47507, P30 CA 36727 andP50CA72712-01.

FIELD OF THE INVENTION

This invention relates to the fields of molecular biology and neoplasticdisease, and more specifically, to isolated nucleic acids, proteins,antibodies, methods and kits containing the same which are useful ingenetic screening assays, and in the design of clinically beneficialchemotherapeutic agents which inhibit the aberrant cellularproliferation in tumor cells.

BACKGROUND OF THE INVENTION

Several publications are referenced in this application by author nameand year of publication in parentheses in order to more fully describethe state of the art to which this invention pertains. The disclosure ofeach of these publications is incorporated by reference herein.

Pancreatic cancer is the fifth leading cause of death by cancer in theUnited States. Twenty-four thousand people die each year from thisdisease. The 5-year survival for pancreatic cancer patients is less than5% and the incidence of the disease has tripled over the last 40 years.The molecular basis underlying the pathogenesis of pancreaticadenocarcinoma remains unknown. As a result, the disease has anextremely poor prognosis and lacks early diagnostic and therapeuticmodalities.

Normal cellular proliferation is finely regulated by the expression ofgrowth-promoting proto-oncogenes and growth-controlling anti-oncogenes.Mutations, rearrangements, deletions, or amplifications that potentiatethe activities of proto-oncogenes result in tumor formation. Similarevents that inactivate anti-oncogenes or tumor suppressor genes disrupttheir role in the cell as negative regulators of cell growth andproliferation.

Gene amplification has been implicated as a common mechanism by whichtumor cells acquire a chemotherapy resistant phenotype. Someamplification units arise at the site of the normal gene, but disperseinto the cytoplasm as double minutes (DMs). These DMs may becomereincorporated and reamplified as homogeneously staining regions (HSRs)or abnormal banding regions (ABRs) at other sites in the genome. DMs andHSRs may be alternate forms of amplified DNA. The DMs are not inheritedstably during cell division because of the lack of centromeres.Integration of DMs into a chromosome is thought to result in theformation of HSRs, which represent a more stable, form of the amplifiedDNA which is maintained as the cell divides. The mechanisms underlyingthis process are not completely understood, but appear to be based onrecombination and unequal distribution of the amplified DNA intodaughter cells.

Cytogenetic amplification has been observed in 8 of 63 primarypancreatic adenocarcinomas analyzed for the presence of DMs. Theexpression of epidermal growth factor, epidermal growth factor receptor,transforming growth factor, erbB-2, erbB-3, and c-met are elevated inpancreatic cancer (Barton et al., 1991; Korc et al., 1992; Lemoine etal., 1992; Prat et al., 1991).

Using restriction landmark genomic scanning (RLGS), Miwa et al (1996)identified a locus at chromosome 19ql3.1-ql3.2 including the AKT2 genewhich was amplified in 20% of pancreatic cancer. Over expression of theAKT2 gene was further shown to be associated with the malignantphenotype of a subset of human ductal pancreatic cancers (Cheng et al.,1996).

No well-defined differentiation pathway has been shown in pancreaticadenocarcinomas, and the biology of this tumor type is generally poorlyunderstood. The present inventors have appreciated a need for theisolation of essential components involved in the regulation ofdifferentiation and proliferation of pancreatic tumor cells. Molecularelucidation of these components will provide novel targets for thedevelopment of antiproliferative and diagnostic agents for cancertreatment and diagnosis.

SUMMARY OF THE INVENTION

This invention provides novel, biological molecules useful foridentification, detection, and/or molecular characterization ofcomponents involved in the regulation of cellular differentiation andtumorigenesis. According to one aspect of the invention, an isolatednucleic acid molecule is provided which includes an isolated openreading frame encoding a phosphoprotein of a size between about 60 to 70kilodaltons. The encoded protein, referred to herein as PD2, comprises atripartite domain structure including a nuclear transport signal, ahelix-loop-helix domain, a nucleotide binding site, and several putativephosphorylation sites.

In a preferred embodiment of the invention, an isolated nucleic acidmolecule is provided that includes a DNA encoding a human PD2 protein.In a particularly preferred embodiment, the human PD2 protein has anamino acid sequence the same as SEQ ID NO:2. An exemplary PD2 nucleicacid molecule of the invention comprises SEQ ID NO:1.

According to another aspect of the present invention, an isolatednucleic acid molecule is provided, which has a sequence selected fromthe group consisting of: (1) SEQ ID NO:1; (2) a sequence specificallyhybridizing with preselected portions or all of the complementary strandof SEQ ID NO:1; (3) a sequence comprising preselected portions of SEQ IDNO:1, (4) a sequence encoding part or all of a polypeptide having aminoacid SEQ ID NO:2. Such partial sequences are useful as probes toidentify and isolate homologues of the PD2 gene of the invention.Accordingly, isolated nucleic acid sequences encoding natural allelicvariants of the nucleic acids of SEQ ID NO:1 are also contemplated to bewithin the scope of the present invention. The term natural allelicvariants will be defined hereinbelow.

According to another aspect of the present invention, isolated human PD2protein is provided. PD2 is a phosphoprotein with a deduced molecularweight of between about 60 kDa and 70 kDa. PD2 comprises a tripartitedomain structure including a nuclear transport signal, ahelix-loop-helix domain, a cyclic AMP or related nucleotide bindingsite, and several putative phosphorylation sites. The expression of thisPD2 protein correlates with the deregulated growth of highlyundifferentiated pancreatic adenocarcinomas.

In a preferred embodiment of the invention, the protein is of humanorigin, and has the amino acid sequence of SEQ ID NO:2. In a furtherembodiment the protein may be encoded by natural allelic variants of SEQID NO:1. Inasmuch as certain amino acid variations may be present inhuman PD2 protein encoded by natural allelic variants, such proteins arealso contemplated to be within the scope of the invention.

According to another aspect of the present invention, antibodiesimmunologically specific for the human PD2 protein described hereinaboveare provided.

Host cells comprising the PD2 encoding nucleic acids of the inventionare also contemplated to be within the scope of the present invention.Such host cells include but are not limited to bacterial cells,mammalian cells, insect cells, fungal cells, and plant cells. ThePD2-encoding nucleic acids may be conveniently cloned into a plasmid orretroviral vector for introduction into host cells. Such cells areuseful in screening methods to identify compounds which regulate and/orinhibit PD2 expression. Compounds so identified may have therapeuticvalue in the treatment of pancreatic cancer.

The present invention also encompasses transgenic mice expressing thePD2 encoding nucleic acids of the invention. The PD2 encoding DNA may bealtered to include any of the following, mutations, alterations,deletions, insertions. In another embodiment, PD2 knockout mice may begenerated to assess the contribution of the PD2 gene to growth anddevelopment.

This invention also provides methods for genetic screening anddiagnostic evaluation of patients at risk for, or currently sufferingfrom, cancer of the pancreas. The hybridization specificity of thenucleic acids of the invention may be used for differential evaluationof patients presenting with phenotypic characteristics common topancreatic cancer. The nucleic acid molecules of the invention can beused as diagnostic hybridization probes or as primers for diagnostic PCRanalysis for PD2 or mutations thereof. Additionally, antisense moleculeswhich may be useful in the regulation of PD2 expression are providedherein. Other methods encompassed by the present invention includeimmunodetection methods for assessing biological samples for thepresence of PD2 proteins.

In another aspect of the present invention, kits are provided forpracticing the methods set forth above. An exemplary kit for screeningtumor samples for PD2 expression includes for example, suitable primersfor PCR amplification of target PD2 sequences. Exemplary primers includethose having the sequence of SEQ ID NOS: 12 and 13. A kit in accordancewith the invention may also contain vials, buffers, a target PD2sequence as a positive control and a protocol sheet. Another exemplarykit may employ immunological methodology. Kits of this type includeimmobilized PD2 protein and antibodies immunologically specific for PD2.Such kits may be used in for immunological assessment of biopsyspecimens for identification and/or quantification of PD2 in pancreatictissues.

The term “isolated nucleic acid” is sometimes used with reference tonucleic acids of the invention. This term, when applied to DNA, refersto a DNA molecule that is separated from sequences with which it isimmediately contiguous (in the 5′ and 3′ directions) in the naturallyoccurring genome of the organism from which it originates. For example,the “isolated nucleic acid” may comprise a DNA or cDNA molecule insertedinto a vector, such as a plasmid or virus vector, or integrated into thegenomic DNA of a prokaryote or eukaryote.

When used with reference to RNA molecules of the invention, the term“isolated nucleic acid” primarily refers to an RNA molecule encoded byan isolated DNA molecule as defined above. Alternatively, the term mayrefer to an RNA molecule that has been sufficiently separated from RNAmolecules with which it would be associated in its natural state (i.e.,in cells or tissues), such that it exists in a substantially pure form.

The terms “isolated protein” or “isolated and purified protein” aresometimes used herein to refer to a protein produced by expression of anisolated nucleic acid molecule of the invention. Alternatively, theseterms may refer to a protein which has been sufficiently separated fromother proteins with which it would naturally be associated, so as toexist in substantially pure form.

The term “substantially pure” refers to a preparation comprising atleast 50–60% by weight of the compound of interest (e.g., nucleic acid,oligonucleotide, protein, etc.). More preferably, the preparationcomprises at least 75% by weight, and most preferably 90–99% by weight,the compound of interest. Purity is measured by methods appropriate forthe compound of interest (e.g. chromatographic methods, agarose orpolyacrylamide gel electrophoresis, HPLC analysis, and the like).

With respect to antibodies of the invention, the term “immunologicallyspecific” refers to antibodies that bind to one or more epitopes of aprotein of interest (e.g., PD2), but which do not substantiallyrecognize and bind other molecules in a sample containing a mixedpopulation of antigenic biological molecules.

With respect to nucleic acids and oligonucleotides, the term“specifically hybridizing” refers to the association between twosingle-stranded nucleotide molecules of sufficiently complementarysequence to permit such hybridization under pre-determined conditionsgenerally used in the art (sometimes termed “substantiallycomplementary”). When used in reference to a double stranded nucleicacid, this term is intended to signify that the double stranded nucleicacid has been subjected to denaturing conditions, as is well known tothose of skill in the art. In particular, the term refers tohybridization of an oligonucleotide with a substantially complementarysequence contained within a single-stranded DNA or RNA molecule of theinvention, to the substantial exclusion of hybridization of theoligonucleotide with single-stranded nucleic acids of non-complementarysequence.

The nucleic acids, proteins, and antibodies, of the present inventionmay be used to advantage diagnostic reagents and tools for assessing themalignant potential of pancreatic adenocarcinomas. They may also be usedas targets for the development of novel chemotherapeutic agents thatregulate differentiation and/or inhibit aberrant cellular proliferationin tumor cells. The transgenic mice of the invention provide a means toassess the function of PD2 in vivo.

The human PD2 molecules described are above may also be used as researchtools and will facilitate the elucidation of the genetic and proteininteractions involved in the regulation of cell division,differentiation, and neoplastic transformation. Methods and kitsemploying such molecules are also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show autoradiographs of Northern blot analysis of RNAisolated from differentiated and undifferentiated pancreatic cell lines.Total RNA (20 μg; lanes 1 and 2) or poly(A+) RNA (lanes 3 and 4) wasseparated by gel electrophoresis and transferred to a nitrocellulosemembrane. Lanes 1 and 3 contained RNA from the differentiated pancreaticcell line HPAF/CD-11. Lanes 2 and 4 contained RNA from the highlyundifferentiated pancreatic cell line Panc 1. FIG. 1A shows thehybridization of a ³²P-labelled PD2 cDNA probe. PD2 is expressed at30-fold higher levels in Panc 1 cells as compared to HPAF/CD-11 cells.FIG. 1B shows an autoradiogram of the same blot hybridized with a³²P-labelled β-actin probe confirming equivalent loading of RNA perlane.

FIG. 2 depicts the nucleotide (SEQ ID NO:1) and deduced amino acid (SEQID NO:2) sequence of PD2 cDNA. The nucleotide sequence is numbered inthe left-hand margin. The deduced amino acid sequence is numbered in theright-hand margin. The polyadenylation signal is underlined.

FIG. 3 depicts the in vitro transcription/translation product of PD2cDNA. RNA transcripts were generated from pBluescript using T7 RNApolymerase and translated in rabbit reticulocyte lysate in the presenceof ³⁵S methionine. The protein products were separated bySDS-polyacrylamide gel electrophoresis and visualized byautoradiography. The DNA constructs used to generate thetranscription/translation products or the negative controls were asfollows: lane 1, PD2 cDNA inserted in pBluescript in the senseorientation; lane 2, PD2 cDNA inserted in pBluescript in the antisenseorientation; lane 3, pBluescript linearized with HindIII; Lane 4,control rabbit reticulocyte lysate containing no exogenous transcripts;Lane 5, construct containing cDNA for PD-1 in the sense orientation.

FIGS. 4A and 4B schematically represent the PD2 primary amino acidsequence, from amino acid 1 through 531. FIG. 4A reveals the relativepositions of three putative protein motifs: a helix-loop-helix domain, anuclear localization domain, and an Arg-rich RNA binding/cAMP bindingdomain, indicated by boxes. FIG. 4B uses boxes to depict the regions ofsequence identity between various proteins (listed in Table 1) and thededuced amino acid sequence of PD2 protein.

FIG. 5 shows the alignment of the helix-loop-helix domain of the deducedPD2 amino acid sequence with the helix-loop-helix domain of theDrosophila Hairy protein (20). The top sequence labeled “A.” representsamino acid residues 30–87 of the Drosophila Hairy protein, and thebottom sequence labled “B.” represents amino acid residues 15–77 of thePD2 protein. Note that the amino acid residues within the “loop” regionsare not directly shown or listed in the alignment. Boxed residuesindicate identity between the two sequences. Asterisks. (*) indicateidentity in the PD2 sequence with other members of the helix-loop-helixfamily of proteins (35).

FIG. 6 shows an alignment of PD2 residues with consensus cAMP bindingdomains of the bacterial catabolite activator protein, and theregulatory subunit of the eukaryotic cAMP-dependent protein kinase. RowA shows residues 198–213 of the RIβ isoform of the regulatory type Isubunit of cAMP dependent protein kinase (16). Row B shows residues322–337 of the RIβ isoform of the regulatory type I subunit of cAMPdependent protein kinase (16). Row C shows residues 69–88 of the E. colicatabolite gene activator protein (16). Row D shows residues 322–341 ofthe deduced PD2 sequence (FIG. 2). Identity among the amino acidsequences is indicated with boxes around the identical residues.

FIG. 7 depicts immunoblotting analysis of cytoplasmic and nuclearprotein fractions from Panc 1 and HPAF/CD11 cells. Equal amounts ofcytoplasmic and nuclear protein lysates from each cell line were loadedonto an SDS-PAGE gel and electroblotted onto nitrocellulose. The PD2p2polyclonal antibody reacted with a band of about 70 kd and 3 smallerfragments of approximately 40–45 kd in the nuclear extracts isolatedfrom both Panc 1 and HPAF/CD-11 cells.

FIGS. 8A and 8B are autoradiographs depicting Northern blot analyses oftotal RNA (20 μg/lane) from a panel of pancreatic carcinoma cell lines.Total RNA was fractionated by electrophoresis on 1.2%agarose/formaldehyde gel and transferred to a nitrocellulose membrane.Pancreatic carcinoma cell lines analyzed were as follows: Colo 357 (lane1), Fa-2C (lane 2), Panc 89 (lane 3), Panc 1 (lane 4), Capan 2 (lane 5),HS 766T (lane 6), SW 979 (lane 7), T3M4 (lane 8), HPAF (lane 9), BxPC 3(lane 10), AsPC 1 (lane 11), QGP-1 (lane 12), MiaPaCa (lane 13) andHGC-25 (lane 14). FIG. 8A shows the results obtained followinghybridization with a ³²P_labelled PD2 cDNA probe. FIG. 8B showshybridization results obtained using a ³²P-labelled 1-actin cDNA probeconfirming equivalent loading of RNA per lane.

FIGS. 9A and 9B are autoradiographs depicting Northern blot analysisshowing expression levels of PD2 mRNA in normal and tumor cell lines andtissues. Total RNA (20 μg/lane) was fractionated by electrophoresis on a1.2% agarose/formaldehyde gel and transferred to a nitrocellulosemembrane. The cell lines and tissues examined were: pancreatic tumorcell lines Panc 1 (lane 4) and HPAF (lane 3); breast carcinoma celllines BT 20 (lane 1) and CAMA-1 (lane 2); colon carcinoma cell lines LS180 (lane 6) and Colo 320 (lane 7); normal human pancreas (lane 5); tailportion of pancreas (lane 8); pancreatic tumors (lanes 9, 10 and 11);normal human foreskin fibroblasts (lane 12); and human B lymphocyte cellline NALM (lane 13). FIG. 9A shows hybridization results obtained usinga ³²P-labelled PD2 cDNA probe. FIG. 9B depicts the results obtainedusing a ³²P_labelled β-actin cDNA probe, to confirm equivalent loadingof RNA per lane.

FIG. 10 is an autoradiograph of Southern blot analysis of genomic DNA(10 μg/lane) from the Panc 1 and HPAF/CD11 pancreatic tumor cell lines.After restriction endonuclease digestion, the DNA waselectrophoretically separated on a 0.8% agarose gel and transferred to anitrocellulose membrane by standard methods. Lanes 1, 2 and 3 containedPanc 1 DNA digested with EcoRI, HindIII and BamHI, respectively. Lanes4, 5, and 6 contained HPAF/CD11 DNA digested with EcoRI, HindIII andBamHI, respectively. The blot was probed with a ³²P-labelled PD2 cDNAprobe, revealing the 30-fold amplification of the PD2 gene in Panc 1cells as compared to HPAF/CD11.

FIG. 11 is an autoradiograph showing results from Southern blot analysisof genomic DNA (10 μg/lane) from a panel of tumor cell lines. Afterrestriction endonuclease digestion with EcoRI, the DNA waselectrophoretically separated on a 0.8% agarose gel. Cell lines analyzedincluded: pancreatic cell lines HPAF/CD11 (lane 1), Panc 1 (lane 2),Colo 357: (lane 3), SW 979 (lane 4), Capan-1 (lane 5), T3M4 (lane 6),and Hs 766T (lane 7); breast cancer cell lines: CAMA-1 (lane 8), MCF-7(lane 9), Sk-BR (lane 10). Colon carcinoma cell lines: WiDr (lane 11),Colo 320 (lane 12) and LS 180 (lane 13). The blot was probed with ³²Plabelled PD2 cDNA.

FIG. 12 is an autoradiograph illustrating that the PD2 gene is amplifiedin human pancreatic biopsies and tumor cell lines. Purified DNA (10 μg)was digested with PstI (P) and BglII (B), fractionated on 8% agarose andtransferred to a nylon membrane. The blot was probed with a ³²P-labelledprobe specific for PD2 (upper panel). The same blot was stripped andrehybridized to a control probe to confirm equal loading in each lane.Tumor (T) and normal tissue adjacent to tumor (C). DNA from patientcases 1–4 are shown in lanes 1–16. Tumor DNA isolated from cases 5 and 6are shown in lanes 17–20. Pancl DNA is shown in lanes 21 and 22.

FIG. 13 is an an ethidium bromide stained gel showing the results ofRT-PCR on normal human and adult pancreatic tissues. RT-PCR productsfrom normal fetal and adult pancreas were fractionated on a 1.5% agarosegel. Lanes 1–3, adult pancreas; lanes 4–6, fetal pancreas; M, 100 basepair ladder.

FIG. 14 depicts immunoblot analysis of the epitope-tagged PD2 protein.After transfection of the epitope-tagged PD2 cDNA construct in thepancreatic tumor cells, clones were isolated for G418 resistance.Lysates for three clones were analyzed. Protein bands were resolved bySDS-PAGE, transferred to nitrocellulose and probed with M2 monoclonalantibody which specifically recognizes the flag epitope. C, cytoplasmiclysate; N, nuclear lysate; M, molecular weight marker.

FIG. 15 shows an autoradiogram of a SDS-PAGE gel run under reducingconditions showing phosphorylation of PD2 protein in the presence of ³²Porthophosphate. APR, vector alone; PD2F, APR vector with PD2 Flagsequences; C, cytoplasmic; N, nuclear.

FIG. 16 is an autoradiogram showing the results of Northern analysis oftransfected NIH3T3 cells. Total cellular RNA (10 μg) purified from humancontrol and transfected cells was fractionated on a 1%agarose/formaldehyde gel, blotted, and hybridized with a cDNA probe toPD2.

FIG. 17 is a graph showing the growth curve for PD2 transfected NIH3T3cell lines.

FIG. 18 is a graph showing the kinetics of tumor growth ofPD2(sense)NIH3T3 cells inoculated into BALB/c nu/nu mice.

DETAILED DESCRIPTION OF THE INVENTION

Adenocarcinomas of the pancreatic ducts make up over 95% of pancreaticnonendocrine tumors, although the duct system accounts for a minorproportion of the normal gland. Accurate staging of pancreatic cancer isrequired to evaluate treatment modalities such as surgical resection,radiotherapy and chemotherapy. A universally satisfactory staging systemhas not been devised, although several staging systems have beenintroduced for clinical practice (reviewed in Eskelinen and Lipponen,1992). The grading of pancreatic adenocarcinomas utilizes generallyaccepted principles of glandular differentiation, nuclear size andmitotic activity (Kloppel et al., 1985), but subjectivity in assessmentand heterogeneity are common. Accurate histological classification ofthese tumors accordingly, has prognostic relevance and should aid in theselection of appropriate therapy.

Well-differentiated tumors (grade 1) contain duct like structures withpolarized cells. Moderately differentiated tumors (grade 2) contain lessdifferentiated duct-like and tubular glands. Poorly differentiatedtumors (grade 3) contain pleomorphic structures, poorly-differentiatedglands, minimal mucus production and large nuclei. The mostdifferentiated tumors grow as tubular structures with a common luminalspace, while less differentiated tumors show a loss of cell polarityresulting in secretion into both luminal and interstitial space (Kern etal., 1987). The tumor growth rate of poorly-differentiated tumors istwice that for well differentiated tumors. Median survival times arecorrelated with the tumor grade: patients with poorly-differentiatedtumors survived for a shorter time than did patients withwell-differentiated tumors (Kloppel et al., 1985; Eskelinen andLipponen, 1992).

In an effort to identify differentially expressed or overexpressed genesthat play a role in maintaining distinct morphological differentiationfeatures exhibited by pancreatic adenocarcinoma cells, a cDNA libraryfrom a poorly differentiated human pancreatic tumor cell line, Panc 1(1,2), was screened with single stranded cDNA probes generated from mRNAprepared from Panc 1 and a well-differentiated human pancreatic tumorcell line, HPAF/CD11 (3). One cDNA clone (PD-2) detected an mRNAexpressed at levels 30-fold higher in Panc 1 cells, as compared toHPAF/CD-11 cells. A similar amplification was observed in the gene copynumber by Southern analysis. The present invention provides thenucleotide sequence, deduced amino acid sequence, and chromosomallocation of this previously undescribed cDNA. The availability of thesesequences enables the practice of genetic screening assays forclassifying particular pancreatic tumors as well as providing noveltargets for the development of clinically relevant chemotherapeuticagents.

I. Preparation of Human PD2-Encoding Nucleic Acid Molecules, PD2Proteins, and Antibodies Thereto

A. Nucleic Acid Molecules

Nucleic acid molecules encoding the human PD2 proteins of the inventionmay be prepared by two general methods: (1) synthesis from appropriatenucleotide triphosphates, or (2) isolation from biological sources. Bothmethods utilize protocols well known in the art. The availability ofnucleotide sequence information, such as a cDNA having the sequence ofSEQ ID NO:1 enables preparation of an isolated nucleic acid molecule ofthe invention by oligonucleotide synthesis. Synthetic oligonucleotidesmay be prepared by the phosphoramidite method employed in the AppliedBiosystems 38A DNA Synthesizer or similar devices. The resultantconstruct may be purified according to methods known in the art, such ashigh performance liquid chromatography (HPLC). Long, double-strandedpolynucleotides, such as a DNA molecule of the present invention, mustbe synthesized in stages, due to the size limitations inherent incurrent oligonucleotide synthetic methods. Thus, for example, a 1.9 kbdouble-stranded molecule may be synthesized as several smaller segmentsof appropriate complementarity. Complementary segments thus produced maybe annealed such that each segment possesses appropriate cohesivetermini for attachment of an adjacent segment. Adjacent segments may beligated by annealing cohesive termini in the presence of DNA ligase toconstruct an entire 1.9 kb double-stranded molecule. A synthetic DNAmolecule so constructed may then be cloned and amplified in anappropriate vector.

Nucleic acid sequences encoding the human PD2 protein may be isolatedfrom appropriate biological sources using methods known in the art. In apreferred embodiment, a cDNA clone is isolated from a cDNA expressionlibrary of human origin. In an alternative embodiment, utilizing thesequence information provided by the cDNA sequence, human genomic clonesencoding PD2 proteins may be isolated. Suitable probes for this purposeare derived from sequences within the PD2 cDNA and include the followingsequences:

5′ AGTGACAAGAGTGGCAGTGG 3′ (SEQ ID NO: 3) 5′ GAGGACAGAGGACAGGCCCA 3′(SEQ ID NO: 4) 5′ CACTCGGCCCAGGAGGATGG 3′ (SEQ ID NO: 5)5′ GACAGTGACTGAGTCCCAGG 3′ (SEQ ID NO: 6)

Such probes may be between 15 and 40 nucleotides in length. For probeslonger than those shown above, the additional contiguous nucleotides areprovided within SEQ ID NO:1.

Additionally, cDNA or genomic clones having homology with human PD2 maybe isolated from other species using oligonucleotide probescorresponding to predetermined sequences within the human PD2 encodingnucleic acids.

In accordance with the present invention, nucleic acids having theappropriate level of sequence homology with the protein coding region ofSEQ ID NO:1 may be identified by using hybridization and washingconditions of appropriate stringency. For example, hybridizations may beperformed, according to the method of Sambrook et al., MolecularCloning, Cold Spring Harbor Laboratory (1989), using a hybridizationsolution comprising: 5×SSC, 5× Denhardt's reagent, 1.0% SDS, 100 μg/mldenatured, fragmented salmon sperm DNA, 0.05% sodium pyrophosphate andup to 50% formamide. Hybridization is carried out at 37–42° C. for atleast six hours. Following hybridization, filters are washed as follows:(1) 5 minutes at room temperature in 2×SSC and 1% SDS; (2) 15 minutes atroom temperature in 2×SSC and 0.1% SDS; (3) 30 minutes-1 hour at 37° C.in 1×SSC and 1% SDS; (4) 2 hours at 42–65° C. in 1×SSC and 1% SDS,changing the solution every 30 minutes.

One common formula for calculating the stringency conditions required toachieve hybridization between nucleic acid molecules of a specifiedsequence homology (Sambrook et al., 1989) is as follows:T _(m)=81.5° C.+16.6 Log [Na+]+0.41(% G+C)−0.63 (% formamide)−600/#bp induplex

As an illustration of the above formula, using [Na+]=[0.368] and 50%formamide, with GC content of 42% and an average probe size of 200bases, the T_(m) is 57° C. The T_(m) of a DNA duplex decreases by 1–1.5°C. with every 1% decrease in homology. Thus, targets with greater thanabout 75% sequence identity would be observed using a hybridizationtemperature of 42° C.

Nucleic acids of the present invention may be maintained as DNA in anyconvenient cloning vector. In a preferred embodiment, clones aremaintained in a plasmid cloning/expression vector, such as pBluescript(Stratagene, La Jolla, Calif.), which is propagated in a suitable E.coli host cell.

PD2-encoding nucleic acid molecules of the invention include cDNA,genomic DNA, RNA, and fragments thereof which may be single- ordouble-stranded. Thus, this invention provides oligonucleotides havingsequences capable of hybridizing with at least one sequence of a nucleicacid molecule of the present invention, such as selected segments of thecDNA having SEQ ID NO:1. As mentioned previously, such oligonucleotidesare useful as probes for detecting or isolating PD2 genes.

Antisense nucleic acid molecules may be targeted to translationinitiation sites and/or splice sites to inhibit the expression of thePD2 gene or production of the PD2 protein of the invention. Suchantisense molecules are typically between 15 and 30 nucleotides inlength and often span the translational start site of PD2 encoding mRNAmolecules. Suitable antisense molecules for controlling the expressionof PD2 are as follows:

5′ CTGGATGGTGGGCGCCATA 3′ (SEQ ID NO: 7) 5′ CCTGGTCCCGCTTTCGTTT 3′ (SEQID NO: 8) 5′ CTAAGGCGGACCCTGGTTT 3′ (SEQ ID NO: 9)

Alternatively, antisense constructs may be generated which contain theentire PD2 cDNA in reverse orientation. Such antisense constructs areexemplified herein.

It will be appreciated by persons skilled in the art that variants(e.g., allelic variants) of PD2 sequences exist in the human population,and must be taken into account when designing and/or utilizingoligonucleotides of the invention. Accordingly, it is within the scopeof the present invention to encompass such variants, with respect to thePD2 sequences disclosed herein or the oligonucleotides targeted tospecific locations on the respective genes or RNA transcripts.Accordingly, the term “natural allelic variants” is used herein to referto various specific nucleotide sequences of the invention and variantsthereof that would occur in a human population. The usage of differentwobble codons and genetic polymorphisms which give rise to conservativeor neutral amino acid substitutions in the encoded protein are examplesof such variants. Additionally, the term “substantially complementary”refers to oligonucleotide sequences that may not be perfectly matched toa target sequence, but such mismatches do not materially affect theability of the oligonucleotide to hybridize with its target sequenceunder the conditions described.

B. Proteins

Full-length human PD2 protein of the present invention may be preparedin a variety of ways, according to known methods. The protein may bepurified from appropriate sources, e.g., transformed bacterial or animalcultured cells or tissues, by immunoaffinity purification. However, thisis not a preferred method due to the low amount of protein likely to bepresent in a given cell type at any time. The availability of nucleicacid molecules encoding PD2 protein enables production of the proteinusing in vitro expression methods known in the art. For example, a cDNAor gene may be cloned into an appropriate in vitro transcription vector,such as pSP64 or pSP65 for in vitro transcription, followed by cell-freetranslation in a suitable cell-free translation system, such as wheatgerm or rabbit reticulocyte lysates. In vitro transcription andtranslation systems are commercially available, e.g., from PromegaBiotech, Madison, Wis. or Gibco-BRL, Gaithersburg, Md.

Alternatively, according to a preferred embodiment, larger quantities ofPD2 protein may be produced by expression in a suitable prokaryotic oreukaryotic system. For example, part or all of a DNA molecule, such as acDNA having SEQ ID NO:1 may be inserted into a plasmid vector adaptedfor expression in a bacterial cell, such as E. coli. Such vectorscomprise the regulatory elements necessary for expression of the DNA inthe host cell positioned in such a manner as to permit expression of theDNA in the host cell. Such regulatory elements required for expressioninclude promoter sequences, transcription initiation sequences and,optionally, enhancer sequences.

The human PD2 protein produced by gene expression in a recombinantprocaryotic or eukaryotic system may be purified according to methodsknown in the art. In a preferred embodiment, a commercially availableexpression/secretion system can be used, whereby the recombinant proteinis expressed and thereafter secreted from the host cell, and readilypurified from the surrounding medium. If expression/secretion vectorsare not used, an alternative approach involves purifying the recombinantprotein by affinity separation, such as by immunological interactionwith antibodies that bind specifically to the recombinant protein ornickel columns for isolation of recombinant proteins tagged with 6–8histidine residues at their N-terminus or C-terminus. Alternative tagsmay comprise the FLAG epitope or the hemagglutinin epitope. Such methodsare commonly used by skilled practitioners.

The human PD2 protein of the invention, prepared by the aforementionedmethods, may be analyzed according to standard procedures. For example,such protein may be subjected to amino acid sequence analysis, accordingto known methods.

The present invention also provides antibodies capable ofimmunospecifically binding to proteins of the invention. Polyclonalantibodies directed toward human PD2 protein may be prepared accordingto standard methods. In a preferred embodiment, monoclonal antibodiesare prepared, which react immunospecifically with the various epitopesof the PD2 protein described herein. Monoclonal antibodies may beprepared according to general methods of Köhler and Milstein, followingstandard protocols. Polyclonal or monoclonal antibodies thatimmunospecifically interact with PD2 protein can be utilized foridentifying and purifying such protein. For example, antibodies may beutilized for affinity separation of proteins with which theyimmunospecifically interact. Antibodies may also be used toimmunoprecipitate proteins from a sample containing a mixture ofproteins and other biological molecules. Other uses of anti-PD2antibodies are described below.

II. Uses of PD2-Encoding Nucleic Acids, PD2 Proteins and AntibodiesThereto

Cellular signalling molecules have received a great deal of attention aspotential prognostic indicators of neoplastic disease and as therapeuticagents to be used for a variety of purposes in cancer chemotherapy. ThePD2 protein of the invention is intimately involved in the regulation ofdifferentiation and neoplastic growth. The biochemical and molecularinteractions of the PD2 gene and protein involved in the genesis andmaintenance of the transformed state and in the maintenance of anundifferentiated state provide novel targets for the development ofchemotherapeutic reagents that may be used to block the growth of tumorcells and/or promote differentiation.

Additionally, PD2 nucleic acids, proteins and antibodies thereto,according to this invention, may be used as research tools to identifyother proteins that are involved in differentiation and transformationprocesses.

A. PD2-Encoding Nucleic Acids

PD2-encoding nucleic acids may be used for a variety of purposes inaccordance with the present invention. PD2-encoding DNA, RNA, orfragments thereof may be used as probes to detect the presence of and/orexpression of genes encoding PD2 proteins. Methods in which PD2-encodingnucleic acids may be utilized as probes for such assays include, but arenot limited to: (1) in situ hybridization; (2) Southern hybridization(3) northern hybridization; and (4) assorted amplification reactionssuch as polymerase chain reactions (PCR).

The PD2-encoding nucleic acids of the invention may also be utilized asprobes to identify related genes from other animal species. As is wellknown in the art, hybridization stringencies may be adjusted to allowhybridization of nucleic acid probes with complementary sequences ofvarying degrees of homology.

Thus, PD2-encoding nucleic acids may be used to advantage to identifyand characterize other genes of varying degrees of relation to the PD2genes of the invention thereby enabling further characterization of theaberrant cell growth associated with pancreatic adenocarcinomas.Additionally, the nucleic acids of the invention may be used to identifygenes encoding proteins that interact with PD2 proteins (e.g., by the“interaction trap” technique), which should further accelerateidentification of the components involved in regulation of cellularproliferation.

Nucleic acid molecules, or fragments thereof, encoding PD2 genes mayalso be utilized to control the production of PD2 proteins, therebyregulating the amount of protein available to participate in themaintenance of deregulated cell growth. As mentioned above, antisenseoligonucleotides corresponding to essential processing sites inPD2-encoding mRNA molecules may be utilized to inhibit PD2 proteinproduction in targeted cells. Alterations in the physiological amount ofPD2 proteins may dramatically affect the activity of other proteinfactors involved in the regulation of cell division.

The PD2 nucleic acids of the invention may be introduced into hostcells. In a preferred embodiment, mammalian cell lines are provided withcomprise a PD2-encoding nucleic acid or a variant thereof. Host cellscontemplated for use include, but are not limited to NIH3T3, CHO, HELA,yeast, bacteria, insect and plant cells. The PD2 encoding nucleic acidsmay be operably linked to appropriate regulatory expression elementssuitable for the particular host cell to be utilized. Methods forintroducing nucleic acids into host cells are well known in the art.Such methods include, but are not limited to, transfection,transformation, calcium phosphate precipitation, electroporation andlipofection.

The host cells described above may be used as screening tools toidentify compounds which modulate PD2 activity. Modulation of PD2activity may be assessed by measuring alterations in PD2 phosphorylationin the presence of the test compound. The morphology of PD2 expressingcells may also be altered in the presence of the test compound. Finally,test compounds will be assessed for the induction of certain pancreaticdifferentiation markers, such as MUC-1 and carbonic anhydrase.

The availability of PD2 encoding nucleic acids enables the production ofstrains of laboratory mice carrying part or all of the PD2 gene ormutated sequences thereof, in single or amplified copies. Such mice mayprovide an in vivo model for cancer, and may be particularly useful instudying pancreatic cancer. Alternatively, the human PD2 nucleic acidsequence information provided herein enables the cloning of the murinehomolog for use in the production of knockout mice in which theendogenous gene encoding PD2 has been specifically inactivated. Methodsof introducing transgenes and knockouts in laboratory mice are known tothose of skill in the art. Three common methods include: 1. integrationof retroviral vectors encoding the foreign gene of interest into anearly embryo; 2. injection of DNA into the pronucleus of a newlyfertilized egg; and 3. the incorporation of genetically manipulatedembryonic stem cells into an early embryo. Production of the transgenicand knockout mice described above will facilitate the molecularelucidation of the role PD2 proteins play in differentiation andtumorigenesis.

The alterations to the PD2 gene envisioned herein include modifications,deletions, and substitutions. Modifications and deletions render thenaturally occurring gene nonfunctional, producing a “knock out” animal.Substitutions of the naturally occurring gene for a gene from a secondspecies results in an animal which produces an PD2 gene from the secondspecies. Substitution of the naturally occurring gene for a gene havinga mutation results in an animal with a mutated PD2 protein. A transgenicmouse carrying the human PD2 gene is generated by direct replacement ofthe mouse PD2 gene with the human gene. These transgenic animals arevaluable for use in vivo assays for elucidation of other medicaldisorders associated with cellular activities modulated by PD2 genes. Atransgenic animal carrying a “knock out” of a PD2 encoding nucleic acidis useful for the establishment of a nonhuman model for pancreaticcancer involving PD2 regulation.

As a means to define the role that PD2 plays in mammalian systems, micecan be generated that cannot make PD2 proteins because of a targetedmutational disruption of a PD2 gene.

The term “animal” is used herein to include all vertebrate animals,except humans. It also includes an individual animal in all stages ofdevelopment, including embryonic and fetal stages. A “transgenic animal”is any animal containing one or more cells bearing genetic informationaltered or received, directly or indirectly, by deliberate geneticmanipulation at the subcellular level, such as by targeted recombinationor microinjection or infection with recombinant virus. The term“transgenic animal” is not meant to encompass classical cross-breedingor in vitro fertilization, but rather is meant to encompass animals inwhich one or more cells are altered by or receive a recombinant DNAmolecule. This molecule may be specifically targeted to defined geneticlocus, be randomly integrated within a chromosome, or it may beextrachromosomally replicating DNA. The term “germ cell line transgenicanimal” refers to a transgenic animal in which the genetic alteration orgenetic information was introduced into a germ line cell, therebyconferring the ability to transfer the genetic information to offspring.If such offspring in fact, possess some or all of that alteration orgenetic information, then they, too, are transgenic animals.

The alteration or genetic information may be foreign to the species ofanimal to which the recipient belongs, or foreign only to the particularindividual recipient, or may be genetic information already possessed bythe recipient. In the last case, the altered or introduced gene may beexpressed differently than the native gene.

The altered PD2 gene generally should not fully encode the same PD2protein native to the host animal and its expression product should bealtered to a minor or great degree, or absent altogether. However, it isconceivable that a more modestly modified PD2 gene will fall within thecompass of the present invention if it is a specific alteration.

The DNA used for altering a target gene may be obtained by a widevariety of techniques that include, but are not limited to, isolationfrom genomic sources, preparation of cDNAs from isolated mRNA templates,direct synthesis, or a combination thereof.

A preferred type of target cell for transgene introduction is theembryonal stem cell (ES). ES cells may be obtained from pre-implantationembryos cultured in vitro. Transgenes can be efficiently introduced intothe ES cells by standard techniques such as DNA transfection or byretrovirus-mediated transduction. The resultant transformed ES cells canthereafter be combined with blastocysts from a non-human animal. Theintroduced ES cells thereafter colonize the embryo and contribute to thegerm line of the resulting chimeric animal.

One approach to the problem of determining the contributions ofindividual genes and their expression products is to use isolated PD2genes to selectively inactivate the wild-type gene in totipotent EScells (such as those described above) and then generate transgenic mice.The use of gene-targeted ES cells in the generation of gene-targetedtransgenic mice is known in the art.

Techniques are available to inactivate or alter any genetic region to amutation desired by using targeted homologous recombination to insertspecific changes into chromosomal alleles. However, in comparison withhomologous extrachromosomal recombination, which occurs at a frequencyapproaching 100%, homologous plasmid-chromosome recombination wasoriginally reported to only be detected at frequencies between 10⁻⁶ and10⁻³. Nonhomologous plasmid-chromosome interactions are more frequentoccurring at levels 10⁵-fold to 10²-fold greater than comparablehomologous insertion.

To overcome this low proportion of targeted recombination in murine EScells, various strategies have been developed to detect or select rarehomologous recombinants. One approach for detecting homologousalteration events uses the polymerase chain reaction (PCR) to screenpools of transformant cells for homologous insertion, followed byscreening of individual clones. Alternatively, a positive geneticselection approach has been developed in which a marker gene isconstructed which will only be active if homologous insertion occurs,allowing these recombinants to be selected directly. One of the mostpowerful approaches developed for selecting homologous recombinants isthe positive-negative selection (PNS) method developed for genes forwhich no direct selection of the alteration exists. The PNS method ismore efficient for targeting genes which are not expressed at highlevels because the marker gene has its own promoter. Non-homologousrecombinants are selected against by using the Herpes Simplex virusthymidine kinase (HSV-TK) gene and selecting against its nonhomologousinsertion with effective herpes drugs such as gancyclovir (GANC) or(1-(2-deoxy-2-fluoro-B-D arabinofluranosyl)-5-iodouracil, (FIAU). Bythis counter selection, the number of homologous recombinants in thesurviving transformants can be increased.

As used herein, a “targeted gene” or “knock-out” is a DNA sequenceintroduced into the germline or a non-human animal by way of humanintervention, including but not limited to, the methods describedherein. The targeted genes of the invention include DNA sequences whichare designed to specifically alter cognate endogenous alleles.

Methods of use for the transgenic mice of the invention are alsoprovided herein. Knockout mice of the invention can be injected withtumor cells or treated with carcinogens to generate carcinomas. Suchmice provide a biological system for assessing the role played by a PD2gene of the invention. Accordingly, therapeutic agents which inhibit theaction of PD2 proteins may be screened in studies using PD2 knock outmice.

As described above, PD2-encoding nucleic acids are also used toadvantage to produce large quantities of substantially pure PD2proteins, or selected portions thereof.

B. PD2 Protein and Antibodies

Purified PD2 protein, or fragments thereof, may be used to producepolyclonal or monoclonal antibodies which also may serve as sensitivedetection reagents for the presence and accumulation of PD2 protein (orcomplexes containing PD2 protein) in mammalian cells. Recombinanttechniques enable expression of fusion proteins containing part or allof PD2 protein. The full length protein or fragments of the protein maybe used to advantage to generate an array of monoclonal antibodiesspecific for various epitopes of PD2 protein, thereby providing evengreater sensitivity for detection of PD2 protein in cells.

Polyclonal or monoclonal antibodies immunologically specific for PD2protein may be used in a variety of assays designed to detect andquantitate the protein. Such assays include, but are not limited to: (1)flow cytometric analysis; (2) immunochemical detection/localization ofPD2 protein in tumor cells or cells in various stages ofdifferentiation; and (3) immunoblot analysis (e.g., dot blot, Westernblot) of extracts from various cells. Additionally, as described above,anti-PD2 antibodies can be used for purification of PD2 protein and anyassociated subunits (e.g., affinity column purification,immunoprecipitation).

From the foregoing discussion, it can be seen that PD2-encoding nucleicacids, PD2 expressing vectors, PD2 protein and anti-PD2 antibodies ofthe invention can be used to detect PD2 gene expression and alter PD2protein accumulation for purposes of assessing the genetic and proteininteractions involved in the control of differentiation andtransformation pathways.

C. Methods of use for the Compositions of the Invention and Kits forPreforming the Disclosed Methods.

From the foregoing discussion, it can be seen that PD2-encoding nucleicacids, PD2-expressing vectors, PD2 proteins and anti-PD2 antibodies ofthe invention can be used to detect PD2 gene expression and alter PD2protein accumulation for purposes of assessing the genetic and proteininteractions involved in malignant transformation of pancreatic cells.

Exemplary approaches for detecting PD2 nucleic acid orpolypeptides/proteins include:

a) comparing the sequence of nucleic acid in the sample with the PD2nucleic acid sequence to determine whether the sample from the patientcontains mutations; or

b) determining the presence, in a sample from a patient, of thepolypeptide encoded by the PD2 gene and, if present, determining whetherthe polypeptide is full length, and/or is mutated, and/or is expressedat the normal level; or

c) using DNA restriction mapping to compare the restriction patternproduced when a restriction enzyme cuts a sample of nucleic acid fromthe patient with the restriction pattern obtained from normal PD2 geneor from known mutations thereof; or,

d) using a specific binding member capable of binding to a PD2 nucleicacid sequence (either normal sequence or known mutated sequence), thespecific binding member comprising nucleic acid hybridizable with thePD2 sequence, or substances comprising an antibody domain withspecificity for a native or mutated PD2 nucleic acid sequence or thepolypeptide encoded by it, the specific binding member being labelled sothat binding of the specific binding member to its binding partner isdetectable; or,

e) using PCR involving one or more primers based on normal or mutatedPD2 gene sequence to screen for normal or mutant PD2 gene in a samplefrom a patient.

A “specific binding pair” comprises a specific binding member (sbm) anda binding partner (bp) which have a particular specificity for eachother and which in normal conditions bind to each other in preference toother molecules. Examples of specific binding pairs are antigens andantibodies, ligands and receptors and complementary nucleotidesequences. The skilled person is aware of many other examples and theydo not need to be listed here. Further, the term “specific binding pair”is also applicable where either or both of the specific binding memberand the binding partner comprise a part of a large molecule. Inembodiments in which the specific binding pair are nucleic acidsequences, they will be of a length to hybridize to each other underconditions of the assay, preferably greater than 10 nucleotides long,more preferably greater than 15 or 20 nucleotides long.

In most embodiments for screening for cancer susceptibility alleles, thePD2 nucleic acid in the sample will initially be amplified, e.g. usingPCR, to increase the amount of the analyte as compared to othersequences present in the sample. This allows the target sequences to bedetected with a high degree of sensitivity if they are present in thesample. This initial step may be avoided by using highly sensitive arraytechniques that are becoming increasingly important in the art.

The identification of the PD2 gene and its association with cancer pavesthe way for aspects of the present invention to provide the use ofmaterials and methods, such as are disclosed and discussed above, forestablishing the presence or absence in a test sample of a variant formof the gene, in particular an allele or variant specifically associatedwith cancer, especially pancreatic cancer. This may be for diagnosing apredisposition of an individual to cancer. It may be for diagnosingcancer of a patient with the disease as being associated with the gene.

In still further embodiments, the present invention concernsimmunodetection methods for binding, purifying, removing, quantifying orotherwise generally detecting biological components. The encodedproteins or peptides of the present invention may be employed to detectantibodies having reactivity therewith, or, alternatively, antibodiesprepared in accordance with the present invention, may be employed todetect the encoded proteins or peptides.

In general, the immunobinding methods include obtaining a samplesuspected of containing a protein, peptide or antibody, and contactingthe sample with an antibody or protein or peptide in accordance with thepresent invention, as the case may be, under conditions effective toallow the formation of immunocomplexes.

The immunobinding methods include methods for detecting or quantifyingthe amount of a reactive component in a sample, which methods requirethe detection or quantitation of any immune complexes formed during thebinding process. Here, one would obtain a sample suspected of containinga PD2 gene encoded protein, peptide or a corresponding antibody, andcontact the sample with an antibody or encoded protein or peptide, asthe case may be, and then detect or quantify the amount of immunecomplexes formed under the specific conditions.

In terms of antigen detection, the biological sample analyzed may be anysample that is suspected of containing the PD2 antigen, such as apancreas or lymph node tissue section or specimen, a homogenized tissueextract, an isolated cell, a cell membrane preparation, separated orpurified forms of any of the above protein-containing compositions, oreven any biological fluid that comes into contact with pancreatictissues, including blood and lymphatic fluid.

Contacting the chosen biological sample with the protein, peptide orantibody under conditions effective and for a period of time sufficientto allow the formation of immune complexes (primary immune complexes) isgenerally a matter of simply adding the composition to the sample andincubating the mixture for a period of time long enough for theantibodies to form immune complexes with, i.e., to bind to, any antigenspresent. After this time, the sample-antibody composition, such as atissue section, ELISA plate, dot blot or Western blot, will generally bewashed to remove any non-specifically bound antibody species, allowingonly those antibodies specifically bound within the primary immunecomplexes to be detected.

In general, the detection of immunocomplex formation is well known inthe art and may be achieved through the application of numerousapproaches. These methods are generally based upon the detection of alabel or marker, such as any radioactive, fluorescent, biological orenzymatic tags or labels of standard use in the art. U.S. patentsconcerning the use of such labels include U.S. Pat. Nos. 3,817,837;3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241,each incorporated herein by reference. Of course, one may findadditional advantages through the use of a secondary binding ligand suchas a second antibody or a biotin/avidin ligand binding arrangement, asis known in the art.

The immunodetection methods of the present invention have evidentutility in the diagnosis of pancreatic cancer. Here, a biological orclinical sample suspected of containing either the encoded protein orpeptide or corresponding antibody is used. However, these embodimentsalso have applications to non-clinical samples, such as in the titeringof antigen or antibody samples, in the selection of hybridomas, and thelike.

In the clinical diagnosis or monitoring of patients with pancreaticcancer, the detection of PD2 antigen, or an increase in the levels ofsuch an antigen, in comparison to the levels in a correspondingbiological sample from a normal subject is indicative of a patient withpancreatic cancer. The basis for such diagnostic methods lies, in part,with the finding that the PD2 nucleic acid identified in the presentinvention is overexpressed in pancreatic cancer tissue samples (seeExamples below). By extension, it may be inferred that this nucleicproduces elevated levels of encoded PD2 proteins which may also be usedas pancreatic cancer markers.

As mentioned previously, cell lines expressing the PD2-encoding nucleicacids or variants thereof may be used in screening methods to identifyagents which modulate PD2 function.

In one broad aspect, the present invention encompasses kits for use indetecting expression of PD2 in pancreatic tissues. Such a kit maycomprise one or more pairs of primers for amplifying nucleic acidscorresponding to the PD2 gene. The kit may further comprise samples oftotal mRNA derived from tissue of various physiological states, such asnormal, early stage and metastatically progressive tumor, for example,to be used as controls. The kit may also comprise buffers, nucleotidebases, and other compositions to be used in hybridization and/oramplification reactions. Each solution or composition may be containedin a vial or bottle and all vials held in close confinement in a box forcommercial sale. Another embodiment of the present invention encompassesa kit for use in detecting pancreatic cancer cells in a biologicalsample comprising oligonucleotide probes effective to bind with highaffinity to PD2 mRNA in a Northern blot assay and containers for each ofthese probes. In a further embodiment, the invention encompasses a kitfor use in detecting PD2 proteins in pancreatic cancer cells comprisingantibodies specific for PD2 proteins encoded by the PD2 nucleic acids ofthe present invention.

The following protocols are provided to facilitate the practice of thepresent invention.

Cell Lines Utilized in Various Analyses

The cell line Panc 1 was obtained from American Type Culture Collection(ATCC), and comprises a poorly differentiated pancreatic adenocarcinomacell line (2). The well differentiated pancreatic tumor cell lineHPAF-CD11 was established at Duke University (3). The sources of otherpancreatic cell lines of various states of morphological differentiationwere: Colo 357, obtained from George Moore (Denver, Colo.); SW 979, Panc89, and QGP-1 from H. Kalthoff (Hamburg, Fed. Rep. of Germany). Thefollowing cell lines were obtained from ATCC: Pancreatic tumor celllines Hs 766T, AsPc-1, BxPc-3, Mia Paca, Capan-1, HGC 25; Human Blymphocyte cell line NALM; NIH3T3 and the SK-MEL-28 melanoma. Humanforeskin fibroblast cells (HUFF) were obtained from primary culturesestablished by Dr. Kay Singer, Duke University Medical Center. Celllines were cultured in Dulbecco's modified Eagle's medium supplementedwith 10% fetal calf serum. Normal pancreata were obtained from DukeUniversity Comprehensive Cancer Center and the University of NebraskaMedical Center tumor and tissue banks using standard procedures.

RNA and DNA Purification

Total cellular RNA from various tumor cell lines was isolated by theguanidine isothiocyanate-cesium chloride cushion ultra centrifugationmethod (4). Cells were washed twice with ice cold phosphate bufferedsaline and lysed with a solution containing 4 M guanidineisothiocyanate, 0.05 M sodium acetate, 250 mM 2-mercaptoethanol. TotalRNA was recovered via sedimentation through a 5.7 M CsCl, 0.025 M sodiumacetate cushion in a Beckman SW 40 Ti rotor centrifuged at 32,000 rpmfor 18 hours. RNA pellets were resuspended in 0.3 M sodium acetate andprecipitated with ethanol. Poly(A+) mRNA was further purified on twocycles of oligo (dT) cellulose affinity chromatography. Genomic DNA fromHPAF/CD-11 and Panc 1 cell lines was purified by the SDS-Proteinase Kdigestion method and then extracted with phenol/chloroform.

Differential Screening and Cloning of PD2 cDNA

The Panc 1 cDNA library was subjected to differential hybridizationusing single stranded cDNA probes made from mRNA of Panc 1 and CD-11cells. The probes were synthesized using mouse mammary leukemia virus(MMLV) reverse transcriptase (BRL, Gaithersburg, Md.) and random hexamerprimers (Pharmacia, Piscataway, N.J.). The reaction was carried out in50 μl of buffer containing 50 mM Tris pH 8.3, 75 mM KCl, 10 mM DTT, 3 mMMgCl, 3 μg random hexamer primers, 200 μM DATP, 20 μM unlabelled dCTP,200 μM dTTP, 40 μCI α-³²P-dCTP, 5 μg actinomycin D, 45 units of RNasin(Promega Biotec, Madison, Wis.), and 300 units of MMLV reversetranscriptase. Following incubation at 37° C. for one hour, the reactionwas stopped by adding EDTA to 20 mM, and fragments larger than 100 bpwere separated by Sephadex G-100 chromatography. RNA in the cDNA-RNAhybrid was hydrolyzed at 65° C. for 30 minutes in an equal volume of 0.6N NaOH and 30 mM EDTA. The specific activity of cDNA obtained was 0.5 to1.5×10⁸ cpm/μg of RNA. For screening, triplicate nitrocellulosemembranes were lifted and subjected to alkaline hydrolysis andneutralization. Prehybridization, hybridization and washing were aspreviously described (5–7). Plaques which hybridized strongly with thePanc 1 cDNA probe, but not with the HPAF/CD-11 cDNA probe, wereselected. The differential reactivity was confirmed through at least twoadditional screening cycles.

Using a DNA insert derived from a differentially expressed cDNA clone,five additional cDNA clones were isolated from a normal human fetalpancreatic cDNA library.

Sequencing Analysis of PD2 cDNA

Single phage plaques selected after differential screening were grown tolarge quantities using either plate lysates or liquid culture followedby glycerol gradient purification (5). EcoRI inserts from purified DNAwere subcloned into pBluescript +/− vectors (pBS) (Stratagene, La Jolla,Calif.). Both single stranded and double stranded templates weresequenced. Single stranded cDNA was prepared using standard techniqueswith the + and − strand pBS phagemids that are hybrid for f1 phage andpBS (Stratagene). Sequencing was performed by specific primer extensionusing Sequenase T4 DNA polymerase under conditions recommended by thesupplier (U.S. Biochemicals, Cleveland, Ohio). The entire cDNA wassequenced twice, in both directions.

In Vitro transcription and Translation of PD2 cDNA

PD2 cDNA was subcloned into pBS (Stratagene) in both orientations at theEcoRI site. The recombinant plasmid was linearized with HindIII,extracted with phenol: chloroform and ethanol precipitated. Linearplasmids containing inserts in both orientations were transcribed usingT7 polymerase following the instructions of the supplier (Promega). Thetranscripts were translated in a rabbit reticulocyte lysate (Promega)using the manufacturer's procedure with 50 μCi of ³⁵S-methionine(Amersham). The products were separated by electrophoresis on either7.5% or 10% SDS polyacrylamide gels, and visualized by autoradiography.Fluorography was used to enhance the radioactive signal using EN3′HANCE(DuPont-NEN).

Northern and Southern Blot Analysis

Total RNA (20 μg) and/or purified poly (A) RNA were fractionated byelectrophoresis on 1.2% agarose gels containing 0.66 M formaldehyde andtransferred to nitrocellulose via capillary blotting. Genomic DNA wasdigested with the indicated restriction enzymes and separated on 0.8%agarose gel electrophoresis. Southern blotting was performed usingstandard procedures (5–7). cDNA probes were labeled with ³²P dCTP usinga random-primed labeling kit (Boehringer Mannheim, Indianapolis, Ind.),and were separated from free label by Sephadex G-50 columnchromatography (Pharmacia). Prehybridization and hybridization for bothNorthern and Southern blots were carried out in a solution of 5×SSPE,50% formamide, 5× Denhardt's reagent, 200 μg/ml of sheared salmon spermDNA and a minimum of 106 cpm/ml of probe at 42° C. for 18 hours. Blotswere washed twice with 2×SSC containing 0.1% SDS at room temperature for15 minutes followed by 4washes with 0.2×SSC, 0.1% SDS at 60° C.

Sequence Analysis and Molecular Modeling

Primary cDNA and deduced amino acid sequence information was analyzedfor homology with previously described molecules using the GCG softwareanalysis program (version 7, Genetics Computer Group, Madison, Wis.),and MacVector 3.5 (International Biotechnologies, Incorporated, NewHaven, Conn.). FASTA searches for similarity were performed using thefollowing databases: GENBANK (75.0), EMBL (33.0) and Swissprot (24.0).

The Molecular Modeling Core Facility at the Eppley Institute, UNMC, wasused for visualizing the crystallographic structure of proteins withhomology to PD2. These included coordinates for aspartateamino-transferase (1AAT, Protein Data Bank, Brookhaven NationalLaboratory) and glutaminyl tRNA synthetase bound to its cognate tRNA(1GSG, Protein Data Bank, Brookhaven National Laboratory). Thestructures were visualized on a Silicon Graphics work station usingSybyl software, developed by Tripos Associates, St. Louis, Mo.

Chromosomal Mapping

The chromosomal localization of the PD2 gene was performed using thegene-based sequence-tagged-site (STS) mapping method as publishedearlier (8). The 3′-untranslated region (UT) of the PD2 cDNA sequencewas used to design primers for PCR screens of both CEPH megabase-insertYAC DNA pools, obtained from Research Genetics, Huntsville, Ala. (9),and Coriell human x rodent somatic cell hybrid DNA pools (10). For PCRamplification, 4 μl of YAC pool DNA was used in a 15 μl reaction with250 μm each DNTP, 100 ng of each primer and 0.4 U of AmpliTaq polymerasein GeneAmp reaction buffer. Reactions were cycled in a Perkin Elmer GeneAmp PCR system 9600 as follows: 4 minutes at 94° C.; then 35 cycles of15 seconds at 94° C., 1 minute 15 seconds at 55° C., 1 minute 15 secondsat 72° C. followed by an extension step of 10 minutes at 72° C. The sameset of primers and conditions were used to screen the somatic cellhybrid panel DNA, NIGMS #1, from Coriell Institute for Medical Research(Camden, N.J.).

Transfection of NIH3T3 Cells with PD2 Sense and Antisense Constructs

The PD2 cDNA was cloned at the EcoRI site of the pcDNA3.1 vector in thesense and antisense orientation. The recombinant constructs weretransfected into NIH3T3 cells using the Lipofectamine method (GIBCO,BRL).

The examples presented below are provided to illustrate certainembodiments of the invention. They are not intended to limit theinvention in any way.

EXAMPLE I Isolation and Sequencing of a cDNA Differentially Expressed inPancreatic Adenocarcinomas

Isolation of cDNA

A cDNA library from a poorly differentiated human pancreatic tumor cellline, Panc 1, was screened for differentially expressed mRNAs usingsingle stranded cDNA probes synthesized from mRNA from thewell-differentiated HPAF-CD11 (3) and poorly differentiated Panc 1 (2)human pancreatic tumor cell lines. Seventeen clones were obtained thathybridized very strongly to the Panc 1 probe and did not hybridize tothe HPAF/CD-11 probe. The characterization of two of these clones haspreviously been published: one encoded the human ribosomal protein S16(2); the other (named PD-1) encoded the human ribosomal protein rpL17(6). The cDNA reported here is named pancreatic differentiation 2 (PD2).A representative Northern blot is shown in FIG. 1A in which the PD2 cDNAinsert hybridized to a 1.9 kb mRNA transcript. A comparison of PD2 mRNAlevels in these cell lines reveals that PD2 is expressed at 30-foldhigher levels in Panc 1 when compared to HPAF/CD-11 as determined bydensitometric analysis. The same filter was probed with a human β-actincDNA as a control for the quality and quantity of mRNA (FIG. 1B). Fiveadditional PD2 cDNA clones were obtained from a normal fetal pancreaticlibrary.

Sequence of the PD2 cDNA

All cDNA clones isolated from the Panc 1 cDNA library and from thenormal fetal pancreatic cDNA library showed 100% identity in sequence.The complete nucleotide sequence and the deduced amino acid sequence ofthe longest PD2 cDNA (1.9 kb) are shown in FIG. 2. The cDNA sequencecontained a 5′ untranslated region of 156 base pairs and a 3′untranslated region of 138 bp. The non-coding 3′ region contained thepolyadenylation signal AATAAA as underlined in FIG. 2. An open readingframe from nucleotide 157 to nucleotide 1752 yields a predictedtranslation product of approximately 60 kDa. A search of the GENBANK andEMBL databases revealed that PD2 is a newly identified sequence, as thenucleotide sequence of PD2 did not show identity to any other depositedsequence.

Characterization of the PD2 cDNA

The PD2 mRNA transcript was expressed using T7 RNA polymerase andtranslated in rabbit reticulocyte lysate in the presence of ³⁵Smethionine. The in vitro translation products were analyzed by 7.5%SDS-PAGE and the results are shown in FIG. 3. When PD2 cDNA was placedin the correct orientation, a protein of approximately 70 kDa was alsoseen along with three other protein bands of about 43 kDa, 44 kDa, and45 kDa (lane 1). This pattern of reactivity was seen when non-reducingSDS-PAGE gels were run on these samples (data not shown). Similarproducts were not produced in lysates that contained linear pBS DNA cutwith Hind III (lane 3), full length PD2 in an antisense orientation,(lane 2) or lysate alone (lane 4). A positive control for in vitrotranscription/translation analysis using the unrelated cDNA clone PD-1(6) showed translation of a protein with the expected size of 17 kDa.The discrepancy between the observed migration of PD2 (70 kDa) andseveral smaller species and the calculated mass (60 kDa) may be due toposttranslational modification in the reticulocyte lysate or intrinsicproperties of the protein. It is unlikely that the different proteinforms are consequences of mutations in the insert that alter the readingframe, or contaminated plasmid preparations, since three independentpreparations from a single colony gave essentially the same pattern(data not shown).

Chromosomal Localization of the PD2 Gene

The PD2 gene was mapped to the short arm of chromosome 19(19p13.11-q11). This location was obtained by linking the YAC that PD2mapped to (CEPH 785_d_(—)8) out four levels to another YAC (CEPH968_g_(—)5). This YAC then had an sequence-tagged-site (STS) (D19S215)mapped to it, which is mapped to Genethon position 0.38 on Chromosome19. This position could not be translated to the cytogenetic map becausea map for chromosome 19 is not yet available for this region. However,the STS (D19S215) has been mapped on the Lawrence Livermore NationalLaboratories physical map of chromosome 19 about 20 Mb from the p-ter.Using the sizes of the four YACs that link PD2 to this STS, a region of19p13.11-q11 was determined.

EXAMPLE II Characterization of the PD2 Protein

Functional Motifs in the PD2 Prot in

A search for protein motif similarities in the GENBANK, EMBL, PDB andSwiss Prot databases, using BLAST and the FASTA program of the GCGsequence analysis software package, revealed that the PD2 protein hassome regions of similarity to several known proteins (Table I).

TABLE I Proteins with partial homology to PD2 ACCESSION POSITION ANDPROTEIN NUMBER* PERCENTAGE IDENTITY Yeast transcriptional P38351277–336, 26% in 60 residues factor PAFI INCENP nuclear protein P53352103–192, 23% in 90 residues Glucokinase pdb/lglk/80 243–280, 36% in 38residues 9431 E. coli valyl tRNA syn- P07118 36–133, 25% in 98 residuesthetase RNA polymerase sigma p2469 68–100, 24% in 33 residues 54 factorYeast ATP dependent P15424 145–159, 47% in 15 residues RNA helicaseYeast Myosin-like Q02455 59–93, 37% in 35 residues protein MLPI E. coliAspartate Amino- P14909 200–270, 27% in 71 residues transferase Simianimmunodefi- P5896, 295–326, 38% in 32 residues ciency virus reverseP5897, transcriptase P19509 P112502 318–364, 24% in 47 residues Murineleukemia virus P11227, 236–271, 27% in 37 residues reverse transcriptaseP03355, P03357 Recombinase flp protein P13784 300–339, 23% in 40residues Trypanosome RNA poly- P16355 313–366, 19% in 54 residues merasecAMP dependent protein PP12849 263–345, 23% in 83 residues kinase, type1 regulatory protein Modification methylase P14571 312–363, 27% in 52residues RSR1 Maternal effect protein P25158 117–176, 26% in 63 residuesoscar (Drosophila) Inclusion body matrix P09524 125–184, 20% in 67residues protein (Viroplasmin) *Accession numbers are from Swissprot (Pand Q), PDB, and EMBL databases, References of each of the sequence isavailable in the databases.

Some of the regions with identity coincide with functionally importantregions of proteins, including a yeast transcriptional factor (11);INCENP nuclear protein (12); Glucokinase (13); valyl-tRNA synthetase(14); aspartate amino transferase (15); the cAMP binding domain of thebacterial Catabolite Activator Protein (16); and eukaryotic regulatorytype I subunit of a cAMP-dependent protein kinase (17). There is alsosimilarity with certain functionally important regions of several otherproteins with known biological activity that are shown in Table I.

The PD2 protein depicted in FIGS. 4A and 4B contains the followingmotifs, a putative nuclear localization signal (KKRK) at residues269–272 (18); a putative arginine rich RNA binding domain (RVRLSKRRAKA;SEQ ID NO: 14) at residues 329–339 with homology to a consensus sequencedescribed in reference (19); and a putative helix-loop helix domain nearthe amino terminus with homology to a family of helix-loop-helix (HLH)proteins described previously (20) that include members of the myc andmyo D families, and several HLH proteins that play important roles inDrosophila development. FIG. 5 shows the alignment of HLH domain of PD2with the HLH domain in Drosophila hairy protein, a negatively actingmember of this family that regulates expression of other genes involvedin segmentation and bristle pattern development (21). Some members ofthis family of proteins contain a basic region important for DNA bindingwhich precedes the first helix. The HLH domain itself has been shown tobe required for heterodimerization of these family members with otherproteins. Some negatively acting HLH proteins that associate with andblock the action of positively acting HLH proteins lack the basic regionpreceding the HLH domain, or contain prolines in their basic region(20). The PD2 protein is similar to the latter of these, in that itcontains prolines and several basic residues in the region immediatelypreceding the putative HLH domain (residues 11–24). See FIG. 5.

Another region of PD2 (residues 263–345, 23% in 83 residues, FIG. 4)demonstrated significant homology with the type I regulatory chain of anintracellular cAMP dependent protein kinase (residues 265–343,) (17).The region of homology fell within one of the two cAMP binding domainsof the regulatory chain of this enzyme. Furthermore, the PD2 amino acidsequence showed significant homology with the conserved residues foundin the cAMP binding site of the E. coli catabolite activator protein(16) and both of the cAMP binding sites found in the regulatory chain ofthe eukaryotic protein kinase, that included critical glutamic acid andarginine residues known to be important in forming hydrogen bondsinvolved in binding the cAMP and forming the pocket around it (16). SeeFIG. 6. The biological relevance of this region of homology with thecAMP binding element is further supported by the fact that anoverlapping sequence (described below) showed homology with an argininerich motif found in several potential RNA binding proteins (19). PD2also contained several phosphorylation sites similar to histone kinaseand casein kinase.

PD2 Prot in in the Cell

To study the production of PD2 protein and examine its localizationwithin the cell, polyclonal antibodies were generated against twosynthetic peptides of the deduced amino acid sequence of PD2. Thepeptides were selected based on their high antigenic index as analyzedby the MacVector Program. PD2 Peptide 1 (PD2 μl) corresponds to aminoacids 142–162 and has the following sequence:

NH₂-RYGISNEKPEVKIGVSVKQQF-COOH (SEQ ID NO: 10)PD2 peptide (PD2p2) corresponds to amino acids 327–348 and has thefollowing sequence:

NH₂-ETRVRLSKRRAKAGVQSGTNAL-COOH (SEQ ID NO: 11)

The peptides listed above were conjugated to KLH and the resultingimmunogen used to immunize rabbits. Serum was then isolated from arabbit immunized with PD2p2 peptide and PD2 antibodies purified usingpeptide affinity chromatography. The antibodies were then used as probesfor Western blotting of cytoplasmic and nuclear extracts from HPAF/CD11and Panc1 cells. The PD2p2 antibody reacted with a protein ofapproximately 70 kd in nuclear extracts from both cell lines. Theintensity of the PD2 protein band in the nuclear extract was 30 foldhigher in Panc1 cells as compared to HPAF/CD11 cells. See FIG. 7. Asimilar level of over expression was seen in PD2 mRNA and gene copynumber in the Panc1 line.

EXAMPLE III Role of PD2 in Differentiation and Tumor ProgressionDifferential Expression of PD2

Expression of the PD2 gene was evaluated further in a panel of 14pancreatic tumor cell lines representing various morphological stages ofdifferentiation. The putative differentiation grade for the varioustumor cell lines was determined from the published morphological andultrastructural descriptions of the cell lines and corresponding tumors(22). Total RNA from these tumor cell lines were fractionated, Northernblotted and probed with PD2 cDNA. The results of this experiment areshown in FIG. 8A. The PD2 cDNA probe hybridized to a message size ofapproximately 1.8 kb in all cell lines, but its expression wassignificantly elevated only in Panc 1. FIG. 8B shows rehybridization ofthe same filter with a β-actin probe confirming equal loading of thegel.

Expression of PD2 mRNA was also evaluated in colon and breast tumor celllines, human foreskin fibroblasts, a Human B lymphocyte cell line andnormal pancreatic tissues. The results, shown in FIG. 9A, revealedrelatively low levels of mRNA transcripts for PD2 in all cell linesexamined except Panc 1. FIG. 9B is a control blot showing equal loadingof RNA.

Differential Amplification of the PD2 Gene

Purified genomic DNA from the poorly differentiated cell line Panc 1 andwell differentiated cell line HPAF/CD 11 were digested with EcoRl, BamHland Hindlll, fractionated by agarose gel electrophoresis, Southernblotted and hybridized to the PD2 cDNA probe as shown in FIG. 10. Theprobe hybridized to two or more fragments raising several possibilities.PD2 could be part of multi-gene family, there may be pseudogenes forPD2, or PD2 is a part of large gene that contains multiple restrictionsites. One of the bands showed a 30-fold amplification in Panc 1 DNA ascompared to HPAF/CD11, suggesting that this corresponds to the geneencoding the transcribed product seen in Panc 1 cells. Furthermore,Southern blot analysis of EcoRl-digested DNA from a large panel of tumorcell lines including six pancreatic tumor cell lines, three breast tumorcell lines and three colon tumor cell lines confirmed that amplificationof the PD2 gene occurred only in the Panc 1 cell line as shown in theFIG. 11.

Tumor specimens were assessed for the amplification of of the PD2 geneas shown in FIG. 12. One tumor biopsy sample also showed amplificationof the PD2 gene. The same blot was stripped and reprobed to confirmequal loading of the lanes. These results demonstrate that PD2 isinvolved in the development of pancreatic cancer.

The expression of the PD2 gene was also assessed in normal human adultand fetal pancreata. The results are shown in FIG. 13. Normal adultpancreas (ages 30–59) showed very low to undetectable levels of PD2expression. However PD2 expression was consistently observed in allfetal tissues examined (age 18–24 weeks). Expression of the PD2 gene infetal but not in adult pancreas further supports a role for PD2 intransformation and differentiation of pancreatic cells.

EXAMPLE IV Expression Construct Analysis of PDZ

Construction of FLAG Epitope-Tagged PD2

A cDNA molecule was prepared which generated a fusion protein comprisingPD2 and the Flag epitope. For this purpose, a double-stranded, syntheticoligonucleotide was designed to encode DYKDDDGSKSAIF which was insertedinto the unique BglII site (bp 1511) of the PD2 cDNA. The in-frameinsertion of the oligonucleotide was verified by sequence analysis.

FIG. 13 shows the results of Western blot analysis confirming expressionand translation of the cDNA fusion construct. The results showed thatepitope tagged PD2 protein is localized mainly in nuclear lysates.

In additional experiments, the 1.9 kb PD2 cDNA was placed under controlof a strong CMV promoter by using the pCDNA3.1 vector for drivingexpression of PD2 in NIH3T3 cells. Stable transfectants were selected ongeneticin (G418). Expression of PD2-specific mRNA in the NIH3T3 cellstransfected with PD2 is shown in FIG. 16. There was no detectable mRNAfor PD2 in the anti-sense PD2 transfected NIH3T3 cells, designatedherein as PD2ASNIH3T3. NIH3T3 cells transfected with a sense construct,designated herein as PD2SNIH3T3, showed the expression of appropriatesize mRNA for the PD2 gene.

Determination of growth kinetics revealed a shorter population doublingtime for PD2SNIH3T3 cells as compared to PD2ASNIH3T3 cells or control,untransfected NIH3T3 cells. Viable transfected cells (5×10³) were platedin triplicate in RPMI medium with 10% fetal calf serum (Day 0). Thefollowing day the medium was changed and cells were harvested in 0.125%trypsin-0.02% EDTA every day and counted. FIG. 17 shows the mean valuesof three dishes over an 8 day time course. The data reveal that NIH3T3cells expressing PD2 in the sense orientation divide more rapidly thancontrol cells not expressing PD2 or cells expressing PD2 in an antisenseorientation.

To investigate transformation properties of PD2SNIH3T3 cells in vivo,BALB/c, nu/nu were inoculated subcutaneously with PD2SNIH3T3,PD2ASNIH3T3 and control NIH3T3 cells. Groups of five mice were used andchallenged with 1×10⁷ cells of each cell type. Mice were palpatedbiweekly over a period of 5 weeks. The time to tumor formation(LP), thenumber of mice developing tumors, the doubling time of the tumors, andthe size of the tumors were noted. Tumor volumes were calculated usingwidth (a) and length (b) measurements (a²×b/2, where a<b). PD2SNIH3T3cells formed tumors in all mice after a latent period of 7 days, whereasno tumores developed in mice receiving identical doses of PD2ASNIH3T3 orNIH3T3 cells over an observation period of five weeks. See FIG. 18.

These results demonstrate that the PD2 gene can transform NIH3T3 cellsand disturb the components that regulate growth rate in transfectedcells.

EXAMPLE V Genetic Testing of Tumor Biopsies for the Presence of PD2

As described in the previous examples, PD2 is overexpressed inpancreatic adenocarcinoma. The availability of the nucleic acids havingthe sequence of Sequence I.D. Nos. 1 as well as the primer sets setforth below provide reagents for genetic testing in patients for thepresence or absence of amplified PD2.

PD2 forward primer: 5′ TTCAGTCAGGCACCAACG 3′ (SEQ ID NO: 12) PD2 reverseprimer: 5′ CGCTGGCCACCCCCATTG 3′ (SEQ ID NO: 13)

DNA may be isolated from biopsy samples. Such procedures are known tothose of skill in the art. The DNA is suspended in 600 microliters of 50mM NaOH in a 1.5 ml eppendorf tube. The tube is then vortexed for 10seconds followed by a 5 minute incubation in a 95 degree hot water bath.Following this incubation, 60 microliters of Tris (1 mM, pH 8.0) isadded to the tube and the sample vortexed for an additional 10 seconds.The tube is centrifuged in a microfuge for 1 minute to pellet the DNA.The supernatant is then discarded and the sample frozen or the DNAprocessed for PCR. Following DNA amplification, the DNA is sequenced inan automated DNA sequencer to confirm the results obtained with PCR.

EXAMPLE VI Generation of a Hamster Model for Pancreatic Carcinogeneis

Little is known about the etiology, pathogenesis and molecular basis ofpancreatic cancer (PC). Deletions, mutations, and rearrangementsnormally activate proto-oncogenes and inactivate anti-oncogenes(tumor-suppressor genes). These genetic alterations culminate inmolecular events leading to deregulation of cell proliferation. Recentstudies have shown that a preferred subset of normal genes is altered inhuman PC biopsies. Although alterations of these genes have been foundin established tumors, nothing is known about early genetic events atthe initiation stage of pancreatic carcinogenesis. Moreover, it isequally unclear whether alteration of one gene is sufficient or whetherseveral simultaneous or a chain of multiple genetic abnormalities isrequired for the initiation of the uncontrolled growth.

It is well established that in the hamster pancreatic cancer model,which mimics the human is disease in many clinical and biologicalaspects, cancer develops not only from ductal/ductular cells but alsofrom within islets, most probably from stem cells. The hormonalabnormalities found in more than 80% of pancreatic cancer patients,including development of diabetes and increased levels of insulin andislet amyloid polypeptide, ubiquitously reflect the involvement of theendocrine pancreas in the pathogenesis of the pancreatic cancer.

We have recently established an in vitro hamster islet culture, in whichthe stepwise cytological, immunohistochemical, cytogenetic and molecularbiological changes could be followed. We have shown that these hamsterpancreatic islet cells can be transformed in vitro by the pancreaticcarcinogen, N-nitrosobis (2-oxopropyl)amine (BOP). After four weeks oftreatment with BOP, the cultured islet cells showed accelerated growthand pleomorphism.

Anchorage independent and in vivo growth was not seen before week 19 oftreatment. The mutation of the K-ras gene at codon 12 (GGT6GAT) wasfound at this stage. This abnormality is consistent with findings inover 90% of human and BOP-induced pancreatic cancer. In vivo,transformed hamster islet cells formed a poorly differentiated invasivecancer. Cytogenetic analysis of transformed hamster cells revealeddeletions of chromosome Y and loss of heterozygosity of chromosomes 7and 11. Hence, this system presents a unique model for elucidatingstepwise early genetic alterations during pancreatic carcinogenesis.

Inasmuch as BOP triggers loss or inactivation of tumor suppressor genes,activation of oncogenes, and/or differential expression oftumor-associated genes in normal pancreatic cells at the initiation ofcarcinogenesis. The genetic alterations caused by these carcinogensappear to be critical in the development of pancreatic cancer.

This hamster model may be manipulated to assess the role of PD2expression in the malignant transformation of the pancreas. Altered PD2encoding nucleic acids may be introduced into these cells and effects ofcell growth rates and morphology assessed. Additionally, the PD2expressing cells may be treated with test compounds to identify thosecompounds which reverse the malignant phenotype. Such compounds may havebeneficial therapeutic value in the treatment of pancreatic carcinomas.

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While certain of the preferred embodiments of the present invention havebeen described and specifically exemplified above, it is not intendedthat the invention be limited to such embodiments. Various modificationsmay be made thereto without departing from the scope and spirit of thepresent invention, as set forth in the following claims.

1. An antibody immunologically specific for an isolated human PD2protein consisting of SEQ ID NO:2, wherein said human PD2 proteincomprises an amino terminal helix-loop-helix domain and a centrallylocalized nuclear transport signal and nucleotide binding site.
 2. Theantibody of claim 1, which is a monoclonal antibody.
 3. The antibody ofclaim 1, which is a polyclonal antibody.
 4. A method for detecting humanPD2 protein consisting of SEQ ID NO: 2, or a fragment thereof in asample, comprising: a) obtaining a sample suspected of containing thehuman PD2 protein or the fragment thereof; b) contacting said samplewith the antibody as claimed in claim 1, under conditions effective toallow the formation of immune complexes; and c) detecting the immunecomplexes so formed.
 5. The method of claim 4, wherein said antibody islinked to a detectable label.
 6. The method of claim 5, wherein saiddetectable label is selected from the group consisting of a radioactive,a fluorescent, a biological, and an enzymatic label.
 7. A kit fordetecting human PD2 protein consisting of SEQ ID NO:2 or a fragmentthereof in a sample, said kit comprising the antibody as claimed inclaim 1 and optionally said PD2 protein for use as a positive controland instructional material.
 8. The kit of claim 7, wherein said antibodyis linked to a detectable label.
 9. The kit of claim 8, wherein saiddetectable label is selected from the group consisting of a radioactive,a fluorescent, a biological, and an enzymatic label.